Nucleic acid sequencing-by-synthesis has the potential to revolutionize the understanding of biological structure and function. Traditional sequencing technologies rely on amplification of sample-based nucleic acids and/or the use of electrophoretic gels in order to obtain sequence information. More recently, single molecule sequencing has been proposed as a way to obtain high-throughput sequence information that is not subject to amplification bias. See, Braslavsky, et al., Proc. Natl. Acad. Sci. USA 100: 3960-64 (2003).
Sequencing-by-synthesis involves the template-dependent addition of nucleotides to a support-bound template/primer duplex. Nucleotides are added to the 3′ end of the primer portion of the duplex by a polymerase. In some cases, the added nucleotides are labeled in a manner such that their incorporation into the primer can be detected. A problem that is encountered when labeled nucleotides are used is that the labels are sometimes undetectable. For example, when fluorescent labels are used they can inadvertently be bleached by exposure to radiation (e.g., light). Also, if labels are reversibly-linked to the nucleotides they can prematurely be cleaved. If the label is prematurely inactivated or removed, incorporation of the affected nucleotide in the sequencing reaction may not be detectable. This results in errors in sequence determination due to these “missing bases” not being detected (i.e., nucleotides that do incorporate but are not detected and recorded due to problems with label detection). The problem of inadvertent label deactivation is a particular problem when optically-detectable labels, such as fluorophores, are used. Fluorophores can be deactivated by bleaching, precleavage (chemical removal prior to a detection step), or by steric inactivation. The present invention solves the problem of undetectable nucleotides in a sequencing-by-synthesis reaction.